1. Field of the Invention
This invention concerns new acetylating and tritioacetylating reagents. In particular this invention concerns acetylating reagent N-acetoxynaphthalimide and tritioacetylating reagents N-tritioacetoxyphthalimide, N-tritioacetoxysuccinimide, and N-tritioacetoxynaphthalimide, as well as processes for their preparation. These reagents are useful for preparation of nonlabelled acetylated compounds and for preparation of acetylated organic compounds radiolabelled with high tritium content. The invention also concerns a method for synthesis of nonlabelled acetylated and tritioacetylated organic compounds from precursors containing a free --NH.sub.2, --SH or --OH group.
2. Background and Related Disclosures
Acetylated and particularly tritiolabelled acetylated organic compounds with high radiolabel content are in high demand. However, their availability and cost depends on available methods for their preparation. While both the acetylating reagents and tritiolabelled compounds are currently available, their cost is high and, in the case of tritiolabelled compounds, the tritium content is typically rather low, which limits their utility.
Therefore, it would be advantageous to have available new methods and reagents for simplified preparation of acetylated organic compounds and for preparation of acetylated tritiolabelled compounds with high tritium content.
The available methods for preparation of acetylated compounds have been shown to be either relatively non-specific, as these reagents may react with other functional groups present in organic compounds, or require multi-step manipulations, non-ambient incubations, other complex reaction conditions and typically afford low yield. All these conditions lengthen the time of and raise the cost of preparation of acetylated compounds such as acetyl Co-enzyme A, acetylcholine, acetylated amino acids or other acetylated organic compounds.
Exemplary of the problems occurring with preparation of acetylated and tritioacetylated compounds is the preparation of acetyl Co--A. The major routes currently used for the production of acetyl Co--A are enzymatic systems or systems utilizing specialty usually high cost reagents. The most commonly used enzyme for the synthesis of acetyl Co--A is acyl co-enzyme A synthetase (ACS). Additionally, the chemical synthesis of acetyl Co--A may employ acetylating reagents such as thioacetate, acetic anhydride, (J. Am. Chem. Soc., 75:2520 (1952)) sodium acetate, (Anal. Biochem., 176:82 (1989) and ibid, 224:159 (1995)), and S-acylthiocholine iodide (J. Org. Chem., 56:3752 (1991)). The disadvantages of the currently available methods, as described in J. Org. Chem. 56:3752 (1991), include low yield, lengthy preparation, high cost of reagents such as ACS, lack of purity and the contamination of products with lipids present in the enzyme preparations.
Some attempts have been made to rectify these problems. For example, activated imido ester compounds which are highly reactive at 0.degree. C. have been reported as useful N-acylating reagents, as described in J. Am. Chem. Soc., 83:1263 (1961), and ibid, 86:1839 (1964).
N-acetoxyphthalimide was used to prepare N-acetylmuramic acid, as described in J. Org. Chem., 30:448 (1963), and N-iodoacetoxysuccinimide was used for iodoacetylation of N.sup.6 -(6-amino-n-hexyl)adenosine-5'-phosphate, as described in J. Med. Chem., 19:1279 (1976).
The limitations of preparation of tritiated acetylated compounds utilizing tritiated acetic anhydride and tritiated acetic acid as acetylating reagents are well known and, as indicated in Br. J. Nutr., 68:365 (1992), include low tritium content, volatility, and poor chemical selectivity. The tritiated acetyl derivatives produced by the acetylating methods described above can be produced only at a very low specific activity and their usefulness is therefore limited (Biochem. J., 121:737 (1971)).
Attempts to prepare acetylated tritiated compounds include a reaction of [.sup.14 C]phenylalanyl-s-RNA with N-acetoxysuccinimide and N-tritioacetoxysuccinimide, Biochem. Biophys. Res. Commun., 25:17 (1966). N-tritioacetoxysuccinimide, at 0.6 Ci/mmole, was used to tritioacetylate insulin according to Biochem. J., 121:737 (1971); a high specific activity tritioacetyl group was made by iodoacetylation of muramyl dipeptide with N-iodoacetylsuccinimide, and subsequent catalytic tritiodehalogenation (J. Label. Compd. Radiopharm., 20:691 (1983)). As an alternative to acetylation, N-Succinimidyl [2,3-.sup.3 H]propionate at very high specific activity has been used to acylate proteins, as described in Science, 208:303 (1980), but such propionylation resulted in appreciable loss of biological activity, as suggested in J. Biol. Chem., 255:3575 (1980), is ineffective in the presence of stabilizing thiols, as indicated in J. Label. Compd. Radiopharm., 20:277 (1983), or requires long reaction times (ibid, 31:459 (1992)).
Therefore, it would be desirable to develop new and more efficient acetylating and tritioacetylating reagents and processes for preparation of nonlabelled acetylated compounds and tritiolabelled acetylated compounds with high tritium content.
A primary aim of this invention is thus to develop non-volatile, stable and facile acetylating and tritioacetylating reagents having demonstrable utility for synthesis of nonlabelled acetylated compounds and tritioacetylated organic compounds from precursors containing a free --NH.sub.2, --SH or --OH group.
Accordingly, this invention describes a preparation of a novel acetylating reagent, N-acetoxynaphthalimide, useful for preparation of nonlabelled acetylated compounds, and a preparation of tritioacetylating reagents, N-tritioacetoxyphthalimide, N-tritioacetoxysuccinimide and N-tritioacetoxynaphthalimide, having a high tritium content useful for preparation of the tritiolabelled acetylated compounds. The invention also describes methods for preparation of acetylated organic compounds from precursors containing free --NH.sub.2, --SH or --OH groups, including amino acids, peptides, Co--A, choline.
All patents, patent applications and publications cited and referred to herein are hereby incorporated by reference.